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HEPES Buffer (1 M, 7.5 pH) Preparation and Recipe

HEPES buffer is one of the Good's zwitterionic buffer with a pH range of 6.8-8.2. It is commonly used in cell culture medium as its pH is well maintained with changes in carbon dioxide concentration.

To prepare  L of HEPES Buffer (1 M, 7.5 pH):

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Table 1. Required components

ComponentAmountConcentration

HEPES (mw: 238.30 g/mol)238.3 g1 M

Prepare 800 mL of dH2O in a suitable container.

Add 238.3 g of HEPES to the solution.

Adjust solution to desired pH by 10N NaOH.

Add dH2O until volume is 1 L. 于4摄氏度保存。

Procedure

Add 2.38 g of HEPES to an appropriate beaker (100-200 mL beaker in this case).

Add about 80 mL of deionized water to the beaker.

Add a stir bar to the beaker and leave it on a stir plate until completely dissolved (~1 min).

Begin monitoring pH of the solution.  It should be acidic (pH ~5).

Add one NaOH pellet to raise the pH towards 7.4.  It should reach about pH of 7

Once the first pellet is fully dissolved, add a second NaOH pellet if necessary to raise the pH to 7.4.  Monitor carefully, and if the pH approaches 7.3/7.4 before the pellet is fully dissolved, stop the stir plate from spinning the rod and carefully remove the NaOH pellet with a clean spatula.

Note: In my experience, about 1.5 pellets are just the right amount to raise the pH to 7.4, so retrieving the second pellet is necessary for achieving the right pH

If the pH goes too high, lower it back to a pH of 7.4 by carefully adding a little HCl, while monitoring the pH (Caution: wear gloves, eye protection and exercise extreme caution with this acid solution)

Once the pH of the solution is 7.4, add enough deionized water to raise the volume to 100 mL.

Sterile-filter, if possible, and store in the refrigerator for up to 4 months or aliquot and freeze at -20 C for future use.